Cathepsin are a family of enzymes which are part of the papain superfamily of cysteine proteases. Cathepsin, B, H, L, N and S have been described in the literature. Recently, cathepsin K polypeptide and the cDNA encoding such polypeptide were disclosed in U.S. Pat. No. 5,501,969 (called cathepsin O therein). Cathepsin K has been recently expressed, purified, and characterized. Bossard M. J. et al., (1996) J. Biol. Chem. 217, 12517-12524; Drake, F. H. et. at., (1996) J. Biol. Chem. 271, 12511-12516; Bromme, D., et al., (1996) J. Biol. Chem. 271, 2126-2132.
Cathepsin K has been variously denoted as cathepsin O or cathepsin O2 in the literature. The designation cathepsin K is considered to be the more appropriate one.
Cathepsins function in the normal physiological process of protein degradation in animals, including humans, e.g., in the degradation of connective tissue. However, elevated levels of these enzymes in the body can result in pathological conditions leading to disease. Thus, cathepsins have been implicated as causative agents in various disease states, including but not limited to, infections by pneumocystis carinii, trypsanoma cruzi, trypsanoma brucei brucei, and Crithidia fusiculata; as well as in schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy, and the like. See International Publication Number WO 94/04172, published on Mar. 3, 1994, and references cited therein. See also European Patent Application EP 0 603 873 A1, and references cited therein. Two bacterial cysteine proteases from P. gingivallis, called gingipains, have been implicated in the pathogenesis of gingivitis. Potempa, J., et al. (1994) Perspectives in Drug Discovery and Design, 2, 445-458.
Cathepsin K is believed to play a causative role in diseases of excessive bone or cartilage loss. Bone is composed of a protein matrix in which spindle- or plate-shaped crystals of hydroxyaptite are incorporated. Type I collagen represents the major structural protein of bone comprising approximately 90% of the protein matrix. The remaining 10% of matrix is composed of a number of non-collagenous proteins, including osteocalcin, proteoglycans, osteopontin, osteonectin, thrombospondin, fibronectin, and bone sialoprotein. Skeletal bone undergoes remodelling at discrete foci throughout life. These foci, or remodelling units, undergo a cycle consisting of a bone resorption phase followed by a phase of bone replacement.
Bone resorption is carried out by osteoclasts, which are multinuclear cells of hematopoietic lineage. The osteoclasts adhere to the bone surface and form a tight sealing zone, followed by extensive membrane ruffling on their apical (i.e., resorbing) surface. This creates an enclosed extracellular compartment on the bone surface that is acidified by proton pumps in the ruffled membrane, and into which the osteoclast secretes proteolytic enzymes. The low pH of the compartment dissolves hydroxyaptite crystals at the bone surface, while the proteolytic enzymes digest the protein matrix. In this way, a resorption lacuna, or pit, is formed. At the end of this phase of the cycle, osteoclasts lay down a new protein matrix that is subsequently mineralized. In several disease states, such as osteoporosis and Paget's disease, the normal balance between bone resorption and formation is disrupted, and there is a net loss of bone at each cycle. Ultimately, this leads to weakening of the bone and may result in increased fracture risk with minimal trauma.
Several published studies have demonstrated that inhibitors of cysteine proteases are effective at inhibiting osteoclast-medicated bone resorption, and indicate an essential role for a cysteine proteases in bone resorption. For example, Delaisse, et. al., Biochem. J., 1980, 192, 365, disclose a series of proteases inhibitors in a mouse bone organ culture system and suggest that inhibitors of cysteine proteases (e.g., leupeptin, Z-Phe-Ala-CHN.sub.2) prevent bone resorption, while serine proteases inhibitors were ineffective. Delaisse, et. al., Biochem. Biophys. Res. Commun., 1984, 125, 441, disclose that E-64 and leupeptin are also effective at preventing bone resorption in vivo, as measured by acute changes in serum calcium in rats on calcium deficient diets. Lerner, et. al., J. Bone Min. Res., 1992, 7, 433, disclose that cystatin, an endogenous cysteine protease inhibitor, inhibits PTH stimulated bone resorption in mouse calvariae. Other studies, such as by Delaisse, et. at., Bone, 1987, 8, 305, Hill, et. al., J. Cell. Biochem., 1994, 56, 118, and Everts, et. al. J. Cell. Physiol., 1992, 150, 221, also report a correlation between inhibition of cysteine proteases activity and bone resorption. Tezuka, et. at., J. Biol. Chem., 1994, 269, 1106, Inaoka, et. al., Biochem. Biophys. Res. Commun., 1995, 206, 89 and Shi, et. al., FEBS Lett., 1995, 357, 129 disclose that under normal conditions cathepsin K, a cysteine proteases, is abundantly expressed in osteoclasts and may be the major cysteine proteases present in these cells.
The abundant selective expression of cathepsin K in osteoclasts strongly suggests that this enzyme is essential for bone resorption. Thus, selective inhibition of cathepsin K may provide an effective treatment of diseases of excessive bone loss, including, but not limited to, osteoporosis, gingival diseases such as gingivitis and periodontists, Paget's disease, hypercalcemia of malignancy, and metabolic bone disease. Cathepsin K levels have also been demonstrated to be elevated in chondroclasts of osteoarthritic synovium. Thus, selective inhibition of cathepsin K may also be useful for treating diseases of excessive cartilage or matrix degradation, including, but not limited to, osteoarthritis and rheumatoid arthritis. Metastatic neoplastic cells also typically express high levels of proteolytic enzymes that degrade the surrounding matrix. Thus, selective inhibition of cathepsin K may also be useful for treating certain neoplastic diseases.
Several cysteine proteases inhibitors are known. Palmer, (1995) J. Med. Chem., 38, 3193, disclose certain vinyl sulfones which irreversibly inhibit cysteine proteases, such as the cathepsins B, L, S, O2 and cruzain. Other classes of compounds, such as aldehydes, nitriles, .alpha.-ketocarbonyl compounds, halomethyl ketones, diazomethyl ketones, (acyloxy)methyl ketones, ketomethylsulfonium salts and epoxy succinyl compounds have also been reported to inhibit cysteine proteases. See Palmer, id, and references cited therein.
U.S. Pat. No. 4,518,528 discloses peptidyl fluoromethyl ketones as irreversible inhibitors of cysteine proteases. Published International Patent Application No. WO 94/04172, and European Patent Application Nos. EP 0 525 420 A1, Ep 0 603 873 A1, and EP 0 611 756 A2 describe alkoxymethyl and mercaptomethyl ketones which inhibit the cysteine proteases cathepsins B, H and L. International Patent Application No. PCT/US94/08868 and and European Patent Application No. EP 0 623 592 A1 described alkoxymethyl and mercaptomethyl ketones which inhibit the cysteine proteases IL-1.beta. convertase. Alkoxymethyl and mercaptomethyl ketones have also been described as inhibitors of the serine protease kininogenase (International Patent Application No. PCT/GB91/01479).
Azapeptides which are designed to deliver the azaamino acid to the active site of serine proteases, and which possess a good leaving group, are disclosed by Elmore et. at., Biochem. J., 1968, 107, 103, Garker et. al., Biochem. J., 1974, 139, 555, Gray et al., Tetrahedron, 1977, 33, 837, Gupton et al., J. Biol Chem., 1984, 259, 4279, Powers et at. J. Biol Chem., 1984, 259, 4288, and are known to inhibit serine proteases. In addition, J. Med. Chem., 1992, 35, 4279, discloses certain azapeptide esters as cysteine proteases inhibitors.
Antipain and leupeptin are described as reversible inhibitors of cysteine protease in McConnell et. at., J. Med. Chem., 33, 86; and also have been disclosed as inhibitors of serine proteases in Umezawa et. al, 45 Meth. Enzymol. 678. E64 and its synthetic analogs are also well-known cysteine proteases inhibitors (Barrett, Biochem. J., 201, 189, and Grinde, Biochem. Biophys. Acta,, 701, 328).
U.S. Pat. No. 5,142,056 described 1,3-diamido-propanones which inhibit HIV protease. 1,3-diamido-propanones have also been described as analgesic agents (U.S. Pat. Nos. 4,749,792 and 4,638,010).
Certain heterocyclic derivatives of amino acids have been disclosed in the art. For instance, Hamada, et. al., PEPTIDE CHEMISTRY, 1983. Proceedings of the 21st Symposium on Peptide Chemistry (1984), and Boden, et. al., Tet. Lett., 1994, 35, 8271 (1994) disclose thiazole derivatives; and Borg, et. al., 1995, 60, 3112, disclose oxadiazole and triazole derivatives.
The synthesis of azatides (polyaclhydrazides) as peptide mimetics has recently been disclosed by Han and Janda, J. Am. Chem. Soc. 1996, 118, 2539.
Thus, a structurally diverse variety of cysteine proteases inhibitors have been identified. However, these known inhibitors are not considered suitable for use as therapeutic agents in animals, especially humans, because they suffer from various shortcomings. These shortcomings include lack of selectivity, cytotoxicity, poor solubility, and overly rapid plasma clearance. A need therefor exists for methods of treating diseases caused by pathological levels of cysteine proteases, including cathepsins, especially cathepsin K, and for novel inhibitor compounds useful in such methods.
We have now discovered a novel class of hydrazidyl, bid-hydrazidyl and bis-aminomehtyl carbonyl compounds which are proteases inhibitors, most particularly of cathepsin K.